Inu incubation system controller

Cells were plated in a 4 well glass bottom chamber slide (Cellvis; Mountain View, CA, USA) pre-coated with 1 µg/mL fibronectin, at a density of 10,000 cells/well. After 2 h of equilibration, the chamber was placed on the Olympus IX83 microscope in a stable environment at 37 °C, 90% humidity and 5% CO₂ using the STXG Stage top Incubator (Tokai Hit Stage top Incubator) connected to an INU incubation system controller (Tokai Hit; Shizuoka-ken, Japan) and imaged for 20 min to 5 h, depending on the experiment. Cells were imaged every 15 s to 5 min, again depending on the experiment, using relief contrast or DIC and/or LED-based fluorescence (X-cite 120 LED Boost, Excilitas Technologies, Waltham, MA, USA with a dry 20×, 40×- or 100× oil immersion objectives.

Epifluorescence: Epifluorescence of fixed images was done at room temperature on an Olympus IX83 outfitted with an X-cite 120 LED Boost light source (Excelitas Tech.) for fluorescent imaging. A Hammamatsu digital camera (C13440-20CU) was used to capture images. 10x, 20x, or 100x objectives were used during epifluorescence experiments, depending on experimental needs. Time-lapse video microscopy: Time-lapse video microscopy was conducted on either the Olympus IX83 or BioTEK Cytation, as noted previously in the methods. For the IX83, an INU incubation system controller from Tokai Hit was used to maintain cells in a stable environment at 37°C, 90% humidity, and 5% CO₂. A 20X objective was employed during time lapse imaging. A BioTEK Cytation 5 was used primarily for adhesion assays. This setup includes a wide field of view camera, high performance imaging controller and a laser autofocus add-on. Imaging can be done at 4X, 10X, or 20X magnification. Confocal: A Zeiss LSM 700 confocal microscope housed at the USUHS Biological Instrumentation Center (BIC) was employed for higher resolution imaging of fixed samples using a 40X oil immersion objective. A Zeiss 980 Airyscan housed at the USUHS BIC was employed for GFP-myosin IIA imaging using a 63X oil immersion objective. All confocal and Airyscan imaging was done with fluorescent filter sets appropriate for each fluorophore.